anti hcov 229e spike  (R&D Systems)

Journal: BioDesign Research

Article Title: Engineered RNA-based activation system for coronavirus sensing in live cells

doi: 10.1016/j.bidere.2025.100040

Figure Lengend Snippet: Fig. 1. The design of coronavirus VERAS RNAs. (a) A scheme depicting the structure of genomic and subgenomic RNAs of 229E and the viral RNA's discontinuous synthesis strategy. (b) Design of the positive-stranded VERAS (+) and proposed activities of VERAS-regulated synthetic genes and host genes, with and without viral infection. (c) Design of the negative stranded VERAS (−) and proposed activation of transgene expression upon viral infection. (d) Design of the VERAS by mimicking the viral genomic and subgenomic RNAs.

Article Snippet: Cells were stained overnight at 4 ◦C with mouse J2 anti-dsRNA (SCICONS #10010200) and rabbit anti-HCoV 229E spike (The Native Antigen Company, #PAB21477–500) primary antibodies diluted 1:250 in BB.

Techniques: Infection, Activation Assay, Expressing

Journal: BioDesign Research

Article Title: Engineered RNA-based activation system for coronavirus sensing in live cells

doi: 10.1016/j.bidere.2025.100040

Figure Lengend Snippet: Fig. 2. Coronavirus infection induces VERAS-mediated reporter expression. (a–d) GFP expression from VERAS (+) (a–b) and VERAS (−) (c–d) with and without 229E infection. GFP intensity is the integrated signal measured using the IncuCyte Live-cell Imaging System. 4 independent biological replicates were performed (4 images were taken for a and c, and 8 images for b and d per biological replicate). Bar represents mean of each group, and each dot represents an individual image. Scale bar in (b) and (d), 400 μm. (e) Immunofluorescence imaging of 293T/hAPN cells transfected with VERASs and infected with 229E. TagBFP marks transfected cells; dsRNA and spike protein co-stained. 3 independent biological replicates were performed, with 8–12 images taken per replicate. Scale bar, 20 μm. (f) GFP intensity in VERAS transfected cells, comparing 229E vs. mock infected conditions. (g) Mander's correlation coefficients between dsRNA or 229E spike vs. GFP signal in cells infected with 229E comparing transfection with VERASs and untransfected control. (h) GFP percent positivity of cells stained positive for 229E spike or dsRNA, comparing 229E vs. mock infected conditions. Data are presented as mean ± s.e.m. P values were calculated by two-tailed Student's t tests. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Source data and P values are provided.

Article Snippet: Cells were stained overnight at 4 ◦C with mouse J2 anti-dsRNA (SCICONS #10010200) and rabbit anti-HCoV 229E spike (The Native Antigen Company, #PAB21477–500) primary antibodies diluted 1:250 in BB.

Techniques: Infection, Expressing, Live Cell Imaging, Immunofluorescence, Imaging, Transfection, Staining, Control, Two Tailed Test

Journal: BioDesign Research

Article Title: Engineered RNA-based activation system for coronavirus sensing in live cells

doi: 10.1016/j.bidere.2025.100040

Figure Lengend Snippet: Fig. 3. VERASs are transcribed and replicated during coronavirus infection. (a–b) GFP expression dynamics in 293T/hAPN cells transfected with in vitro transcribed VERAS-3 and -6 (positive strand, a) or VERAS-3 (negative strand, b) following 229E infection. (c) RT-PCR-based detection of (+) and (−) RNA transcripts from VERASs. (d) Gel image of RT-PCR products for detection of the (−) RNA transcribed from (+) VERAS at 24 hpi. (e) Replication of VERAS-3 (+) and VERAS-6 (+) during 229E infection. Cells were transfected with the in vitro transcribed RNAs, infected with 229E, and total cellular RNA was collected at 1, 10, 22, and 36 hpi. VERAS RNA levels were quantified by RT-qPCR and normalized to 1 hpi. Data represents 3 biological replicates (3 technical repeats each), shown as mean ± s.e.m. Statistical significance determined by two-tailed Student's t-test: n.s., not significant; *P < 0.05; ***P < 0.001. Source data and P values are provided.

Article Snippet: Cells were stained overnight at 4 ◦C with mouse J2 anti-dsRNA (SCICONS #10010200) and rabbit anti-HCoV 229E spike (The Native Antigen Company, #PAB21477–500) primary antibodies diluted 1:250 in BB.

Techniques: Infection, Expressing, Transfection, In Vitro, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

Journal: BioDesign Research

Article Title: Engineered RNA-based activation system for coronavirus sensing in live cells

doi: 10.1016/j.bidere.2025.100040

Figure Lengend Snippet: Fig. 4. VERASs encode additional proteins and are packaged into progeny virions for cell transmission. (a) Positive-strand genomic and subgenomic VERAS designs encoding both GFP and mRuby3. (b–d) GFP and mRuby3 expression in the 293T/hAPN cells with or without 229E infection at 48 hpi. Bars: means; circles: technical replicates (n = 4 biological replicates, 8 images each). Scale bar, 200 μm. (e) Negative strand bicistronic VERAS designs. (f–g) Flow cytometry quanti fication of GFP and mRuby3 expression from VERAS (−) (n = 3 biological replicates, 3 technical replicates each). (h) SARS-CoV-2 (S), 229E (E), and OC43 (O) VERASs with packaging sequences. (i) GFP signal in cells re-infected with virions from VERAS-transfected (S, E, O) or control (NC) cells, mock vs 229E or OC43 infection. Bars: means; circles: individual images (n = 4 biological replicates, 4 images each). Statistical significance: two-tailed t-test (b–d, f–g); one-tailed t-test (i); n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Source data available.

Article Snippet: Cells were stained overnight at 4 ◦C with mouse J2 anti-dsRNA (SCICONS #10010200) and rabbit anti-HCoV 229E spike (The Native Antigen Company, #PAB21477–500) primary antibodies diluted 1:250 in BB.

Techniques: Transmission Assay, Expressing, Infection, Flow Cytometry, Transfection, Control, Two Tailed Test, One-tailed Test

Journal: BioDesign Research

Article Title: Engineered RNA-based activation system for coronavirus sensing in live cells

doi: 10.1016/j.bidere.2025.100040

Figure Lengend Snippet: Fig. 5. VERASs for live-cell virus detection and infection-activated antiviral. (a) GFP intensity in 293T/hAPN cells infected with or without 229E virus at various loads. Data shown as box plots (median, 25–75th percentiles, min–max) from 4 biological replicates (8 technical repeats each). (b) Apoptosis signal (Annexin V) in cells transfected with or without VERASs encoding apoptosis inducers (Bax or Caspase3) and infected with 229E. Data from 4 biological (8 images each). (c) 229E virus titers in media from cells transfected with or without a VERAS expressing IFNα subtypes (positive or negative strand) and infected with 229E. Data from 3 biological replicates (3 technical repeats each), shown as mean (bar) and individual data points (circles). (d) Death signal (Cytotox green dye) of the infected cells. Data from 3 biological replicates (16 images each), presented as violin plots. (e) Scheme of co-culture assay to assess protective effects of VERAS-3 (+) or VERAS-3 (−) IFNA8 on neighboring cells. (f) Death rate of GFP+ cells in total GFP+ cells. One-tailed Student's t-tests for p values. (g) Mechanism and future applications of VERAS system that harnesses viral regulatory machineries to detect virus infection and trigger antiviral therapy. Data from 3 biological replicates, shown as mean ± s.e.m. Statistical Analysis: P values calculated by two-tailed Student's t-test unless otherwise noted. NC, no transfection negative control; n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Source data provided.

Article Snippet: Cells were stained overnight at 4 ◦C with mouse J2 anti-dsRNA (SCICONS #10010200) and rabbit anti-HCoV 229E spike (The Native Antigen Company, #PAB21477–500) primary antibodies diluted 1:250 in BB.

Techniques: Virus, Infection, Transfection, Expressing, Co-culture Assay, One-tailed Test, Two Tailed Test, Negative Control